Circularly Permutated Haloalkane Transferase Fusion Molecules

This application is a Continuation-In-Part of U.S. patent application Ser. No. 17/604,417, filed on Oct. 17, 2021, which is the U.S. National Stage of International Patent Application No. PCT/EP2020/060785 filed on Apr. 16, 2020, which claims priority to European Patent Application No. 19169689.7 filed on Apr. 16, 2019, and European Patent Application No. 19206641.3 filed on Oct. 31, 2019.

The Sequence Listing is submitted as an XML file named 95083_381_2001_seq created Jul. 28, 2025, about 17,000 Bytes, which is incorporated by reference herein in its entirety.

The present invention relates to polypeptide sequences comprising a haloalkane transferase enzymatic activity, which are capable of modulating this enzymatic activity in response to an environmental stimulus.

Methods for integrating biochemical processes over time are of great scientific interest. These biochemical processes encompass, for example, protein-protein interactions, changes in metabolite concentration or protein sub-cellular (re)localization. Currently, these biochemical processes are mainly studied employing real time fluorescence microscopy using tailor-made biosensors. However, these approaches suffer from several drawbacks. Notably in neurobiology, experimental setups required for fluorescence imaging in vivo restrict animal movement and affect the studied behaviours. Biochemical processes occurring in organs such as the intestines or the heart are complex to resolve at the cellular level due to their inherent physiological movements. More generally, fluorescence microscopy is restricted to confined fields of view and tissue depth. Despite great improvements in equipment, image processing and biosensor designs, it remains impossible to study fundamental events in complete rodent organs in vivo by fluorescence microscopy.

An alternative approach consists in the recording of biochemical processes that can be read out at a later time point. This recording process is defined as integration. It leads to an irreversible mark being accumulated over time in response to the biochemical process under investigation.

The post hoc evaluation of experiments is not only valuable for in vivo studies, but can also be beneficial for cell-based assays. Especially in the case of rarely occurring biochemical processes, the signal integration over time can improve the readout due to an enhanced signal to noise ratio. In high throughput screenings, signal integration could offer a snapshot of the studied phenomenon, thereby increasing multiplexing and reducing costs by replacing lengthy recordings via real-time microscopy. Finally, using fluorescent substrates, cell populations can be identified, and eventually sorted, based on their metabolic/signalling profile for downstream analysis and/or treatments.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for integrating biochemical processes over time. This objective is attained by the subject-matter of the independent claims of the present specification.

Based on the self-labelling protein HaloTag™ 7 (PDB 6Y7A), the inventors provide a chemogenetic integrator family of proteins that have been engineered to irreversibly react with chloroalkane substrates in response to a given biochemical process. These integrators consist of split circular permutants of HaloTag™ 7 (PDB 6Y7A) that change conformation in response to a biochemical process and invoke labelling activity. In a typical experiment, the recording window is defined by the time presence of the chloroalkane substrate that can (covalently) label the chemogenetic integrator. Different labelling substrates can be employed sequentially in pulse-chase experiments offering the possibility to study different successive phenomena with the same biological sample.

The invention relates to fusion proteins comprising a circularly permutated haloalkane transferase split into two parts, which are catalytically active when brought into close spatial proximity by a sensor polypeptide module but are otherwise catalytically inactive.

The nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

HaloTag™ 7 (see GenBank AQS79242); the cp version employed in creating the invention does not contain the C-terminal 27 amino acids of this sequence.

Specifically, the invention relates to a modular polypeptide system comprising a first partial effector sequence comprising

A second aspect of the invention relates to nucleic acids encoding the fusion protein of the invention. Alternatively, nucleic acids are provided that encode the two parts of the circularly permutated haloalkane transferase (the first partial effector sequence and the second partial effector sequence). These nucleic acid sequences encoding the first and second partial effectors are useful for making other fusion proteins capable of sensing analyte concentrations or protein-protein interactions with yet unexplored interaction partners or sensor modules.

Furthermore, the invention provides expression systems, cells and transgenic non-human animals comprising the fusion proteins or encoding nucleic acids of the invention. Similarly, kits providing the nucleic acids for rapid construction of transgenic expression constructs and suitable substrate compounds are encompassed by the invention.

The term fluorescent dye in the context of the present specification relates to a small molecule capable of fluorescence in the visible or near infrared spectrum. Examples for fluorescent labels or labels presenting a visible colour include, without being restricted to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanine (APC), peridinin chlorophyll (PerCP), phycoerithrin (PE), Alexa Fluors™ (Life Technologies™, Carlsbad, CA, USA), DYLIGHT™ fluors™ (Thermo Fisher™ Scientific, Waltham, MA, USA) ATTO Dyes (ATTO-TEC GmbH, Siegen, Germany), BODIPY™ Dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene based dyes) and the like.

Amino acid sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3ed. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof.

The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15 amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.

In the context of the present specifications the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

The term having substantially the same activity in the context of the present specification relates to the activity of an effector polypeptide pair, particularly SEQ ID NO: 004 and SEQ ID NO: 007 (PEP2), i.e. haloalkane transferase activity. A polypeptide qualified as having substantially the same activity does not necessarily show the same quantity of activity as the reference polypeptide; in the particular case of the present invention, a reduction of enzymatic turnover with respect to the reference peptide cpHalo might indeed be desirable for certain applications. As laid out below, for purposes of distinguishing polypeptides covered by the present inventions from those that are not covered, the inventors propose a threshold of activity of 10sMin the standard assay as laid out in Example 9, with Halo-CPY as the substrate.

For purposes wherein the above definition of activity is not applicable, 3 standard deviations above background with regard to haloalkane transferase activity shall be taken as the reference threshold for having substantially the same activity. In certain embodiments, at least 5 standard deviations are used as the reference threshold. In certain particular embodiments, at least 10 standard deviations are used as the reference threshold.

In the context of the present specification, the term amino acid linker refers to a polypeptide of variable length that is used to connect two polypeptides in order to generate a single chain polypeptide. Unless specified otherwise, exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids.

In the context of the present specification, the term Gltl is an abbreviation for bacterial periplasmic glutamate binding protein (Uni-Prot-ID: H4UFY3).

A first aspect of the invention relates to a method for detecting a specific molecular interaction between two proteins or peptides. These interaction partners are further referred to herein as a first sensor polypeptide and a second sensor polypeptide, and each of the sensor polypeptides is part of a complex with a part of an effector polypeptide sequence.

The first sensor polypeptide is covalently attached through a peptide bond to a first partial effector sequence. This first partial effector sequence comprises or consists essentially of

The first sensor polypeptide and the first partial effector polypeptide together constituting a first interaction complex.

The second sensor polypeptide is covalently attached to a second partial effector sequence comprising or consisting essentially of a sequence selected from SEQ ID NO: 006 (PEP1), SEQ ID NO: 007 (PEP2) and a sequence at least (≥) 75% identical to SEQ ID NO: 007 (PEP2).

The second sensor polypeptide and the second partial effector sequence together constitute a second interaction complex.

In certain embodiments, said sequence at least (≥) 75% identical to SEQ ID NO: 007 (PEP2) has at least one mutation at position A151, R146, E147, T148, or T154 with respect to SEQ ID NO: 007 (PEP2).

The first and second partial effector sequences together constitute a circularly permuted haloalkane dehalogenase, and are capable, when brought into close proximity of each other, to effect covalent attachment of a halogen alkane moiety.

The method according to the invention comprises the steps of: contacting said first sensor polypeptide and said second sensor polypeptide in the presence of a haloalkane dehalogenase substrate, and determining whether covalent attachment of said haloalkane dehalogenase substrate to said first partial effector sequence has occurred, thereby detecting specific molecular interaction between said first sensor polypeptide and said second sensor polypeptide.

The first and second interaction complexes each contain one of the two sensor partners, the interaction of which is interrogated by the method of the invention. In the event of an association of the two sensor polypeptide partners, the effector sequences comprised in the interaction complexes re-constitute an active haloalkane dehalogenase, which will then covalently attach a haloalkane dehalogenase substrate to the first effector sequence part of the interaction pair. Concurrent or later detection of this covalent attachment of the haloalkane dehalogenase substrate then testifies that the two partners have interacted.

Detection of the haloalkane dehalogenase substrate attachment is advantageously performed by detecting or utilizing a label that the haloalkane dehalogenase substrate bears covalently attached to it.

In certain embodiments, the haloalkane dehalogenase substrate is covalently attached to a fluorescent dye moiety. A variety of methods are known to the skilled artisan to assert presence of absence of the dye molecule, including real-time spectroscopic methods such as fluorescence depolarization or fluorescence microscopy.

Fluorescence depolarization, also referred to as fluorescence anisotropy, enables real-time monitoring of the attachment of a fluorescent dye to a substrate by measuring changes in the rotational mobility of the fluorophore. Upon excitation with polarized light, the emitted fluorescence retains a degree of polarization that is inversely related to the rate of molecular rotation during the excited-state lifetime. When a fluorescent dye is free in solution, it rotates rapidly, resulting in low anisotropy; upon binding to a larger substrate or surface, its rotational mobility decreases, leading to an increase in fluorescence anisotropy. By detecting changes in anisotropy over time, the binding kinetics or conjugation of the fluorescent dye to the substrate can be continuously monitored.

In particular embodiments, the label is a fluorescent dye moiety and determining whether covalent attachment of said haloalkane dehalogenase substrate to said first partial effector sequence has occurred is performed by determining a fluorescence signal.

In certain embodiments, the haloalkane dehalogenase substrate is covalently attached to an affinity tag moiety.

The term “affinity tag” in the context of the present specification refers to a small molecule (MW<1000 Da) or peptide. The affinity tag is capable of specifically interacting with a corresponding binding partner to form a covalent or high-affinity (k<10mol/L) non-covalent interaction, thereby enabling selective separation, immobilization, or detection of the tagged first partial effector sequence from a solution or complex mixture. Such interactions include, but are not limited to, biotin-streptavidin binding, peptide-antibody recognition (for FLAG and related tags), or engineered tag-ligand systems, and may facilitate purification, localization, or labeling of the tagged molecule under physiological or experimental conditions.

In particular embodiments, the label is an affinity tag moiety selected from the group consisting of biotin, a FLAG™, a Strep™-tag, a Glutathione S-transferase (GST) tag, a SNAP tag™ substrate, and a CLIP tag™ substrate. Such substrates are described, inter alia, in U.S. Pat. Nos. 7,939,284 B2 and 8,367,361 B2, incorporated by reference herein.

Biotin binds with exceptionally high affinity (non-covalently) to avidin, streptavidin, or NeutrAvidin™, forming a nearly irreversible complex that enables robust capture and separation. The FLAG™ tag is a short, hydrophilic peptide sequence that binds non-covalently and with high specificity to anti-FLAG antibodies, facilitating immunoaffinity-based detection or purification. The Strep-tag, such as Strep-tag II or Twin-Strep-tag, is a short peptide that binds non-covalently to Strep-Tactin, a modified streptavidin with enhanced affinity, allowing for selective purification under mild elution conditions using desthiobiotin or biotin.

The Glutathione S-transferase (GST) tag is a protein fusion tag that binds non-covalently to immobilized glutathione, enabling affinity purification via glutathione agarose or similar matrices. The SNAP tag is a self-labeling protein tag derived from O-alkylguanine-DNA alkyltransferase that forms a covalent bond with benzylguanine-functionalized ligands, enabling irreversible labeling or immobilization. The CLIP tag, closely related to SNAP, is engineered to covalently react with benzylcytosine derivatives, allowing orthogonal and irreversible labeling in parallel with SNAP-tagged constructs.

In particular embodiments, determining whether covalent attachment of said haloalkane dehalogenase substrate to said first partial effector sequence has occurred is performed by contacting the first partial effector sequence with a surface coated with a binding partner to the affinity tag, and determining the presence of the first partial effector sequence or of the first sensor polypeptide on said surface.

Surfaces that may be employed in this context include microtiter wells and magnetic particles. Presence of the first partial effector sequence may be determined by a range of methods including specific detection ligands (such as antibodies), but also by detecting a fluorescence signal. If the haloalkane dehalogenase substrate attached to the first effector polypeptide bears a fluorescent dye in addition to the affinity tag, capture of the substrate-labelled first interaction complex may be combined with optical detection, enabling massively parallel automated assays of interaction.

In some embodiments, the first partial effector sequence and the second partial effector sequence, when brought into close proximity of each other, comprise an activity of 10sMin a fluorescence polarization assay using N-(10-(2-carboxy-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)phenyl)-7-(dimethylamino)-9,9-dimethylanthracen-2(9H)-ylidene)-N-methylmethanaminium as the substrate.

In some embodiments, the first partial effector sequence and the second partial effector sequence, when brought into close proximity of each other, have at least 0.5%, of the activity of SEQ ID NO: 001.

In some embodiments, the internal linker comprises or consists of the amino acids G, A, J, S, T, P, C, V, M.

In some embodiments, the first partial effector sequence comprises or essentially consists of

The method according to the invention relies on a modular analyte sensor polypeptide system (also named modular polypeptide herein) that comprises, or essentially consists, of a split effector polypeptide sequence having HaloTag™activity, which is reconstituted by a sensor polypeptide module.